The optimal time for a neck ultrasound (US) in the follow-up of papillary thyroid cancer (PTC) after the first year is undetermined. We aimed to verify the utility of routine neck US in the surveillance of patients diagnosed with low- and intermediate-risk PTC with no evidence of disease at the one-year assessment.We conducted a retrospective longitudinal study of patients with low- and intermediate-risk PTC with normal neck US, unstimulated serum thyroglobulin (Tg) < 1 ng/mL and negative anti-Tg antibodies at the one-year follow-up. Patients were divided into group 1 [undetectable Tg (<0.20 ng/mL)] and group 2 [detectable Tg but < 1 ng/mL]. The negative predictive value (NPV) of the one-year unstimulated Tg at the five-year and last follow-up visits was calculated.We included n = 88 patients in group 1 and n = 8 patients in group 2. No patient from group 1 presented suspicious US findings at the five-year evaluation [NPV: 100.0% : 95.5%-100.0%)], and at the last visit, only one patient had developed a lymph node classified as suspicious . In group 2, two patients’ USs presented suspicious findings at the five-year evaluation [NPV: 75.0% (95% CI: 34.9%-96.8%)]. At the last visit, only one patient persisted with suspicious findings in the US .Low- and intermediate-risk PTC with an excellent response to treatment at the one-year assessment can be safely monitored with regular unstimulated Tg assessments. Conclusions should not be drawn for Tg levels between 0.20-0.99 ng/mL. Differentiated thyroid cancer (DTC) represents the most common form of thyroid cancer, and the vast majority of these malignancies exhibit low or intermediate risk of recurrence   - 5 . CoSome authors consider neck US the main assessment tool in the surveillance of DTC due to its high resolution, easy access, non-invasiveness, speed of execution and usefulness in the qualitative characterization of the thyroid parenchyma and respective lymph nodes as well as the detection of recurrence   - 10 . CRecent studies have also shown that the increasing use of neck USs has led to more therapeutic interventions, such as surgeries and radioiodine remnant ablation (RRA), but not with an increase in survival rates   , 11 . MIncreasing evidence supports the performance of clinical assessment, neck US and measurement of unstimulated serum Tg levels at the one-year evaluation of low- and intermediate-risk DTC and, in subsequent years, only a neck US for patients in whom levels of Tg exceed the threshold of 1 ng/mL   , 18 . OThe aim of this study was to assess the utility of routine neck USs in the surveillance of patients diagnosed with low- and intermediate-risk PTC    with neThis was a retrospective longitudinal single-center study conducted at the Endocrinology Department of the Hospital de Braga.The study was approved by the Ethics Committee for Health of the Hospital de Braga and the Ethics Committee for Life and Health Sciences of the University of Minho. For the entire duration of the study, we ensured the patients’ anonymity and confidentiality.® .We reviewed data from patients followed in the Thyroid Group Consultation (TGC) between January 2017 and October 2020 with low- and intermediate-risk PTC, according to the 2015 ATA guidelines ( The inclusion criteria were the following: i) age at diagnosis of 18 years old or higher; ii) histologically confirmed diagnosis of PTC; iii) risk of recurrence classified as low or intermediate, according to the ATA Initial Risk Stratification System ( The exclusion criteria were i) patients with suspicion or evidence of disease and/or TgAb positivity at FUV-1, ii) histological diagnosis of other DTC: follicular or Hürthle cell carcinoma or aggressive DTC variants , and iii) any missing data from the inclusion criteria.All the patients who met the above criteria were included in the study.The selected patients underwent thyroidectomy between 2011 and 2015. Our general surgery department utilized an institutional protocol based on recommendations the American Thyroid Association published in 2009. When the histopathology report confirmed a diagnosis of PTC, the patients were referred to the TGC. The TGC follow-up protocol for PTC consists of an initial assessment in the first 12 months after surgery and subsequent reassessments at least once a year. The follow-up includes® 2000 Thyroglobulin, Siemens Healthcare Diagnostics Inc, functional sensitivity 0.9 ng/mL, analytical sensitivity 0.2 ng/mL);Determination of serum Tg levels ; Cobas® Elecsys TSH, Roche Diagnostics GmbH (from 2008 to 2010); Dimension Vista® 1500 System, Siemens Healthcare Diagnostics Inc (from 2011 to 2020); AtellicaTM IM Thyroid Stimulating Hormone (TSH) assay, Siemens Healthcare Diagnostics Inc (since 2020)];Determination of serum TSH levels ; andDetermination of serum TgAb levels . Thirteen patients presented with a Tg-I status and three patients with a Tg-S status   .At the last FUV (mean follow-up time: 6.7 ± 1.2 years), one patient had developed a neck lymph node status classified as US-S, with Tg-N [NPV: 98.8% (95% CI: 93.2% to 100.0%)]. Thirteen patients presented with a Tg-I status and one patient with a Tg-S status   .Applying the composite endpoint to define absence of disease (both serum Tg < 1 ng/mL and US-N status) at the last FUV, we obtained an NPV of 97.5% (95% CI: 91.3% to 99.7%).At the last FUV (mean follow-up time: 6.5 ± 1.8 years), the US-I neck lymph node detected at FUV-5 did not persist. The patient with US-S + Tg-I findings had a total follow-up time of five years; therefore, the data reported at FUV-5 and the final FUV belong to the same FUV. Consequently, a total of seven patients had US-N status [NPV of a Tg-I + US-N status at the FUV-1: 87.5% (95% CI: 47.4% to 99.7%)]. At the final FUV, one patient had developed Tg-S status (with US-N) and two other patients presented with Tg-I.Analysis of the composite endpoint to define absence of disease (serum Tg < 1 ng/mL and US-N status) at the last FUV resulted in an NPV of 75.0% (95% CI: 34.9% to 96.8%).DTC, particularly PTC, is associated with low recurrence and mortality rates   , 10 . NThe main findings of the current study suggest that the follow-up for PTC patients with an excellent response to treatment can be safely based on assessments of unstimulated serum Tg levels without a routine neck US. At the one-year assessment, in patients with US-N, a Tg-N status (excellent response to therapy) – group 1 – strongly predicted the absence of sonographic evidence of cervical lymph node disease at FUV-5 and the last FUV.Before proceeding with further analysis of the results, it is important to mention one important caveat concerning our data. Due to group 2’s small sample size, its results should be interpreted with caution. This limitation resulted in a wide CI and hence a less precise estimated effect. Consequently, we cannot assertively make similar inferences with indeterminate levels of unstimulated Tg (0.20-0.99 ng/mL); therefore, further discussion of results will focus on the group of patients with an excellent response to treatment (group 1). The reports from the group 1 patients’ neck USs only described one lymph node classified as suspicious (in intermediate-risk patients). Its presence would have been missed if assessment were based on Tg and TgAb alone, as none of these values were suspicious. This finding probably corresponds to false positives; nevertheless, no further data is available, as the report corresponds to this specific patient’s last FUV.We also reported one patient with a neck lymph node classified as indeterminate at FUV-5. However, we considered it a false positive finding, as the subsequent neck US was normal. This result is consistent with previous studies that showed high rates of false positives from neck USs   - 14  anOur results support evidence from previous observations reported by Grani and cols.    in a stRegarding previous neck dissection, we had a high percentage of patients submitted to this procedure (58.8%) due to the less conservative approach to neck dissection in the institutional protocol in force at the time based on the 2009 ATA guidelines. Although we recognize that a previous neck dissection might lead to a higher rate of postsurgical athyroglobulinemia, potentially increasing the sensitivity of thyroglobulin for long-term follow-up   - 28 , wSome limitations of our study must be considered. First, because it is a retrospective and single-center study, the power of the results is limited, as they may not be suitable for other populations. Additionally, there is the constraint associated with the small sample size for group 2 (n = 8), precluding conclusions for unstimulated Tg levels between 0.20 and 0.99 ng/mL. Moreover, the results cannot be extrapolated to patients diagnosed with other, more aggressive variants of DTC, as they exhibit atypical clinical and biological characteristics. Nevertheless, the less aggressive and more classic variants addressed in this study (PTC) represent the vast majority of diagnosed DTC   - 34 . TThe strengths of our study include the fact that our findings are consistent with those reported by similar studies conducted in different centers. Furthermore, the TGC consists of a team of physicians with extensive experience in the field, who conduct well characterized and extensive routine assessments, which, combined with strict inclusion and exclusion criteria and a rigorous US-report classification (each reviewed and classified by two investigators), allowed for the inclusion of a more uniform sample and reliable data, however leading to a smaller sample size.In conclusion, our results show that low- and intermediate-risk PTC patients treated with total thyroidectomy with an excellent response to treatment  at the 1-year evaluation can be safely monitored with regular unstimulated serum Tg assessments. In these patients, neck USs should be reserved for those patients with rising serum Tg levels and/or TgAb titers.These results could lead to fewer unnecessary diagnostic procedures and interventions and better quality of life for the patients as well as lower costs in their long-term follow-up. Nevertheless, further prospective studies on this topic should be conducted, and larger samples are necessary to confirm these findings for patients with serum Tg levels between 0.20 and 0.99 ng/mL.


The synergistic effect of immunotherapy and radiotherapy has been widely recognized. However, the cumulative lung toxicity of thoracic radiotherapy (TRT) combined with PD-1/PD-L1 inhibitors cannot be overlooked in clinical practice. In the PACIFIC study, the incidence of pneumonitis was significantly higher in the immunotherapy consolidation group compared to the placebo group (33.9% vs. 24.8%) [The study was approved by the Ethics Committee of Shandong Cancer Hospital. As it was a retrospective analysis, informed consent was not required. We included patients with non-small cell lung cancer (NSCLC) who received TRT and at least 2 cycles of immunotherapy, with the interval between the two treatments not exceeding 6 months. The primary endpoint was treatment-related pulmonary toxicity, assessed using the Common Terminology Criteria for Adverse Events (version 5.0).A total of 148 patients who received thoracic conventional fractionated radiation therapy were included , and the incidence of grade ≥ 2 pneumonitis was 10.3% vs. 32.7%  in the anlotinib-treated group and the non-anlotinib-treated group . Nevertheless, the mechanisms behind this effect still require further investigation.Currently, corticosteroids are commonly used in clinical practice to treat radiation-immunotherapy-related pneumonitis, but they may affect the anti-tumor efficacy of drugs . On the This study offers a new alternative for clinical treatment approaches; however, it necessitates larger prospective studies to explore safety and efficacy aspects further.


Cyberchondria is defined as the increase in health-related anxiety or anguish associated with excessive or repeated online searches for health-related information. Our objective was to cross-culturally adapt and validate the CSS-12 scale for Peruvian Spanish speakers, to determine whether the Bifactor model works as well in our population as in previous studies’ and to explore whether the Bifactor-ESEM is a more suitable model.We performed a cultural adaptation using the Delphi method and a validation study on medical students between 2018 and 2019. Reliability was evaluated by using Cronbach’s alpha (α) and McDonald’s omega (Ω) for internal consistency, and Pearson’s r and intraclass correlation coefficient (ICC), for test-retest reliability. We evaluated construct validity by contrasting four measurement models for the CSS-12 and the convergent validity against health anxiety.The Spanish CSS-12 showed excellent reliability . The Bifactor ESEM model showed the best fit, supporting a unidimensional measure of the general cyberchondria. This measure was positively associated with health anxiety (r = .51).The Spanish CSS-12 provides a valid and reliable unidimensional measure of cyberchondria, which is distinguishable from the more general health anxiety. This can be applied to similar populations and future research. The Bifactor-ESEM model appears to offer a more accurate and realistic representation of the multifaceted nature of cyberchondria. We provide a free-to-use form of the Spanish CSS-12 as supplemental material. The increased distress or anxiety related to repeated or excessive searches online for health-related information is defined as “cyberchondria” (CYB) , 2. CYB High levels of HA associated with increased access to health-related information on the Internet can carry considerable repercussions. For example, it can affect work and social development since too much time is spent investigating possible diseases . FurtherThe Cyberchondria Severity Scale (CSS) is a self-report instrument designed to facilitate this construct’s multidimensional measure . The CSSBased on this evidence, the original authors developed a short version of the CSS -known as the Cyberchondria Severity Scale– 12 (CSS-12)–to overcome the limitations reported by others . In the Although the Bifactor model has demonstrated adequate psychometric properties, testing a Bifactor-ESEM model is relevant given the intriguing patterns within the cyberchondria . For exaThe CSS-12 is a more robust version to evaluate CYB, but it is not available in Spanish. Indeed, there are no instruments for assessing CYB in Spanish-speakers, partially explaining the absence of research about this critical topic in Latin America. Our objective was to cross-culturally adapt and validate the CSS-12 scale for Peruvian Spanish speakers. We also wanted to determine whether the Bifactor model works as well in our population as in previous studies’ and to explore whether the Bifactor-ESEM is a more suitable model.We developed this study in two steps: (a) adaptation to Spanish and (b) psychometric validation. The latter included evaluating the adapted CSS-12 regarding reliability, structure validity and external validity against an independent measure of anxiety.The original version of the CSS-12  was indeWe performed a psychometric validation of the adapted CSS-12 on medical students recruited from a Peruvian university. We selected this population because the original CSS-12 was validated in college students; thus, it facilitates comparisons between both studies. We planned to evaluate at least 360 participants , following standard recommendations for the statistical analysis described below . We initWe evaluated the reliability by assessing the internal consistency of the CSS-12 items using the 657 cross-sectional applications. Cronbach’s alpha and McDonald’s omega were calculated to assess the general scale’s internal consistency and per each domain , 28. AddWe evaluated the construct validity of the CSS-12 and its convergent validity against an indicator of HA. For the latter, we applied the Spanish version of the Short Health Anxiety Inventory (SHAI) , 31. We The CSS-12  uses a LThe Short Health Anxiety Inventory (SHAI) serves to quantify anxiety about health. It consists of 18 questions divided into two sections, 14 questions about the perceived likelihood of becoming ill (Main Section) and four questions corresponding to the perception of the negative consequences of an illness (Negative Consequences Section). Each question presents four options to be scored from 0 to 3, which allow obtaining a total score between 0 and 54 points (a score of 0 to 42 for the Main Section and 0 to 12 for the Section of Negative Consequences). The SHAI has validated versions in Spanish , 31, andFor the adaptation step (a), we calculated medians of the final outcomes from the expert opinion after finishing the Delphi method.We started the psychometric validation (b) by summarising relevant information from each item. We calculated the average and standard deviation (SD) of each item, domain and the general CSS-12 scale. At the item level, we also calculated the inter-item correlation (polychoric correlation matrix), item-total correlation, item-rest correlation, and Cronbach’s alpha conditioned to the item elimination.For assessing reliability, we estimated internal consistency and stability over time, as explained above. We considered Cronbach’s alpha and McDonald’s Omega values around 0.70 as adequate reliability, values around 0.80 as very good reliability, and values around 0.90 as excellent reliability , 34. We For evaluating the structure validity, we compared four CSS-12 measurement models. These models were estimated by using a Weighted Least Square Mean and Variance (WLSMV) estimator and specified as follows:Model-1: A one-dimensional model for which all items loaded in one general factor . This is a confirmatory factor analysis (CFA) model.Model-2: A four-dimension model for which all factors (the four CSS-12 domains) are correlated. The model was specified as follows: EXC loads on items 1, 3, 6; COMP loads on items 2, 7, 10; DIST loads on items 4, 8, 9; and REAS loads on items 5, 11, 12. This is a confirmatory factor analysis (CFA) model.Model-3: A model with four specific factors  and a general factor (CYB), all uncorrelated. The four specific factors were uniquely defined with their respective main loadings , setting all other cross-loadings equal to zero. The general factor was defined through main loadings from all items. This is the Bifactor model evaluated by the CSS-12 creators [creators .Model-4: A model similar to Model 3, but instead of setting all other cross-loadings equal to zero, they are free parameters to estimate. This is a Bifactor-ESEM as described by Morin et al [in et al  and is oΩH) and (ii) explained common variance (ECV) [ΩH reflects the proportion of the total variance attributable to the general CSS-12 factor (CYB) after controlling the effect for the specific factors EXC, COMP, DIST and REAS. Values >.80 imply that CYB is the main source of score variability [As visible, we built our modelling approach based on the original CSS-12 work by McElroy et al. . In our ce (ECV) , 45. ΩH iability . ECV is iability , 48.As explained previously, we evaluated the convergent model validity by calculating the correlation (Pearson’s r) between the SHAI and CSS-12 raw scales. Data management was supported by Microsoft Excel ®  and the statistical analysis by STATA® Version 16.0  and Mplus™ Version 8.4 .The Institutional Review Board of the Universidad Peruana de Ciencias Aplicadas reviewed and approved the study . All participants freely agreed to participate and gave their verbal informed consent after fully understanding the study objectives. All data were handled anonymously, in encrypted devices, and no risks were compromising sensitive information.The Delphi instrument evaluated by the five experts obtained scores ≥ 3 in each of the five aspects evaluated in the first round of review, with the acceptance of the instrument as culturally adapted. The final wording of the items is shown in the supplemental material.Table 1. Polychoric correlation matrix between items of the CSS-12 is available in S1 Table.The CSS-12 total score average was 25.1 (SD 9.1). The average scores for each of the four dimensions were: Excessiveness = 7.8 (S.D. 2.8); Distress = 5.7 (S.D. 2.6); Reassurance = 5.8 (S.D. 2.6); and Compulsion = 5.8 (S.D. 2.5). Item mean ranged from 1.6 (SD 0.9) to 3.0 (SD 1.1), and item-total correlations ranged from 0.58 to 0.76. Item mean, standard deviation, item-total correlations and Cronbach’s alpha if removed are presented in Table 2) and were very good with McDonald’s omega .The CSS-12 total score demonstrated excellent internal consistency , and internal consistency values were in the adequate–very good range for the subscales with Cronbach’s alpha . The global intraclass correlation also revealed very high reliability for the instrument (ICC = 0.93) and domains .The Pearson’s correlation coefficients calculated from the test-retest for both the items and domains showed very high reliability, with all values being positive and >0.80  to the most complex Bifactor-ESEM model (Model 4). CFI, TLI, RMSEA and SRMR also improved their values in a similar way. This empirical evidence supports the most complex Bifactor-ESEM solution. The general CYB factor of the Bifactor-ESEM explains a considerable fraction of the shared variance , allowing to sum the 12 items for obtaining a total score. Standardized factor loadings for this model are visible in Table 5.Concerning construct validity, fit statistics for one-factor CFA, first-order four-factor CFA, Bifactor, and Bifactor-ESEM models of the CSS-12 are presented in Table 2).Regarding convergent validity, the correlation between CSS-12 and SHAI was positive and moderate (Pearson’s r = 0.51), showing that some patients with health anxiety are likely to have cyberchondria as well. Weak to moderate correlation was evident when evaluating each subscale with SHAI . Convergent validity between the English CSS-12 and the SHAI was previously reported , showingWe have identified some limitations and strengths in our study. Given that the CSS-12 tool was only validated in students from a private Peruvian college, the external validity is limited. Although they are healthy in general, this population is commonly linked to “transient hypochondriasis”, which increases the levels of HA that usually occurs in medical students . This coIn conclusion, the Spanish version of the 12-items Cyberchondria Severity Scale (CSS-12) was successfully adapted for the Peruvian college students’ population. It provides a valid and reliable unidimensional measure of the cyberchondria, which is distinguishable from the more general health anxiety. The Bifactor-ESEM model seems to offer a more accurate and realistic representation of the multifaceted nature of cyberchondria, contributing to a comprehensive understanding of this phenomenon. The CSS-12 can be applied for evaluating cyberchondria in similar populations and future research.S1 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


In the age- and sex-matched analysis, the population ratio with a minimum response (≤100 binding antibody units (BAU/mL) was higher among patients with AIH than among controls 26 weeks after the second vaccination . Conclusions: The anti-SARS-CoV-2 spike antibody concentration in AIH patients was significantly lower than that in controls after the second vaccination. Continued and widespread vaccination, particularly for patients requiring medical immunomodulation, is recommended.Background/Aims: Although the World Health Organization declared the end of the public health emergency of international concern focusing on COVID-19 in May 2023, this bothersome virus continues to mutate, and the possibility of the emergence of mutant strains with high infectivity and severe disease rates has not disappeared. Thus, medical evidence must be accumulated, which is indispensable for protecting both patients under immunosuppressive treatments and the healthy population. This study examined SARS-CoV-2 vaccination responses in Japanese patients with autoimmune hepatitis (AIH) compared with healthy controls. Methods: This observational study registered 22 patients with histologically diagnosed AIH and 809 healthy controls in our hospital. Their Elecsys anti-SARS-CoV-2 spike antibody concentrations before and after vaccination were evaluated. Results: In this study, 72.7% and 18.2% of patients with AIH received steroids and azathioprine, respectively. Significant negative correlations were found between age and anti-SARS-CoV-2 spike antibody concentration in both groups; however, no sex differences were found. Although anti-SARS-CoV-2 spike antibody concentration was drastically augmented after the second vaccination ( The incidence of autoimmune hepatitis (AIH), a chronic inflammatory liver disease with unknown etiology, is increasing worldwide ,2, and wAccording to the World Health Organization (WHO), as of June 2023, more than 750 million infections and approximately 7 million deaths have been reported worldwide with respect to COVID-19 . On the The first mRNA vaccine in human history, which was rapidly developed in response to the COVID-19 pandemic, is attracting attention as a useful tool to avoid severe COVID-19 infection . HoweverIn this study, we examined SARS-CoV-2 vaccination responses in Japanese patients with AIH and compared them with the results of healthy medical workers using propensity score-matched analyses. ® Anti-SARS-CoV-2 , cases with antibody titers of 1.0 or higher were considered positive. Finally, 809 healthy controls were enrolled in this study (control group).In this study, 22 Japanese patients with histologically diagnosed AIH who attended our hospital between May 2021 and March 2022 based on the international AIH criteria suggested by the International Autoimmune Hepatitis Group  were volAll study participants were vaccinated with BNT162b2 mRNA COVID-19 vaccine ©. General information about healthy controls, patients, and types and doses of immunosuppressants were collected. In the control group, blood sampling was performed before the first vaccination and 12/26 weeks after the second vaccination. In the AIH group, blood sampling was performed before the first vaccination, 3 weeks after the first vaccination, and 4/12/26 weeks after the second vaccination.® Anti-SARS-CoV-2 S and the other was Elecsys® Anti-SARS-CoV-2 . The former is for the quantitative determination of antibodies to the SARS-CoV-2 spike protein and is useful not for assessing past SARS-CoV-2 infection but for assessing the effect of SARS-CoV-2 vaccination. The latter is for the quantitative determination of antibodies to the SARS-CoV-2 nucleocapsid protein and is useful not for assessing the effect of SARS-CoV-2 vaccination but for assessing the past SARS-CoV-2 infection. Serum concentrations of these antibodies were measured in our laboratory using the electrochemiluminescence immunoassay method.Two different types of antibodies for SARS-CoV-2 were used in this study. One was Elecsysp < 0.05 were considered statistically significant. JMP version 14.3  was used for statistical analyses.Numerical variables were expressed as medians and quartiles. The Mann–Whitney U test was adopted to examine the significant difference between two groups without a normal distribution. Unrelated categorical variables were evaluated by using Pearson’s chi-square test. Correlation was assessed using Spearman’s rank correlation coefficients. Values of https://www.mhlw.go.jp/content/000769923.pdf. accessed on 30 June 2023]. This study was conducted according to the Declaration of Helsinki, and informed consent was obtained via an opt-out method. The Ethical Committee of Nara Prefecture General Medical Center and Nara Medical University approved this study , which was conducted according to ethical principles and Japanese ethics guideline for life science and medical research involving human subjects [p < 0.01). In the AIH patient group, some patients who had been diagnosed as AIH in the past and continuously treated for two or three decades were also included in the study. The male-to-female ratio of the AIH group was 4/18, which was not significantly different from that (186/623) of the control group (p = 0.79). In the AIH group, 72.7% (16/22) of the patients were treated with adrenal steroids, and the median daily dose of prednisolone was 5.5 mg. Azathioprine was prescribed in 18.2% (4/22), with a median dose of 75.0 mg per day. None of the patients with AIH received anti-allergic agents. The clinical data of controls were not supplied because not all the participants in the control group had their medical records in our hospital and detailed information on steroids, azathioprine, and anti-allergic drugs were not able to be collected.The clinical profiles of the 809 controls and 22 patients with AIH enrolled in this study are shown in p < 0.001 and p < 0.001, respectively). A similar negative correlation was observed in the AIH group (p < 0.001 and p = 0.209) (As shown in = 0.209) .p < 0.05) and tended to gradually decrease (p = 0.08). As shown in p < 0.005 and p < 0.005, respectively). The changes in humoral vaccination response over time in the AIH group are shown in p < 0.0001; tolerable response: 70% vs. 3.5%, p < 0.0001; p < 0.0001; tolerable response: 73% vs. 10%, p < 0.0001; Given that the predictive degree of immunity to SARS-CoV-2 based on antibody levels has not been determined yet , the cutp = 0.86). The male-to-female ratio in the control group was 2/13 and was not significantly different from the value of 3/12 in the AIH group (p = 1.00). In the AIH group, 73.3% (11/15) of the patients were treated with prednisolone, and its median daily dose was 6.0 mg. Immunomodulator was prescribed to 20.0% (3/15) of the patients, and its median dose was 75.0 mg. As shown in p < 0.05). Both endpoints, “poor” and “tolerable” responses, in the AIH patient group occurred more frequently than in the control group 12 weeks after the second vaccination  © was approved in February 2021, and in this study, the anti-SARS-CoV-2 spike antibody concentration after vaccination was markedly lower in the AIH group than in the control group. In addition, after 3 months of repeated vaccinations, the majority of patients already had antibody titers at levels considered clinically inadequate. Patients with AIH are generally ubiquitous among middle-aged and older women, and the continuation of immunomodulating agents such as adrenal steroids and thiopurines is essential for disease control ,22, and Several cases of AIH-like liver injury have been noted after COVID-19 vaccination ,24,25,26This study has several limitations that should be considered when interpreting the results. First, the sample size of patients with AIH was limited because the prevalence of AIH was not high, and in the chaos that followed the spread of COVID-19, recruiting a large number of patients to participate in this study was difficult. Second, the results of this study may be biased by measured and unmeasured confounding factors because this study was retrospectively performed in a hospital in Japan and all participants were Japanese. Third, the control group included regular employees at the hospital. Therefore, although a relatively large number of healthy young or middle-aged women were included, the possibility that they were taking immunomodulatory medications or had malignant diseases was not necessarily ruled out with certainty. Finally, information on antibody titers after the third or fourth vaccination has not been collected.Despite these limitations, we successfully demonstrated that the anti-SARS-CoV-2 spike antibody concentration in the AIH group was significantly lower than that in the control group until 6 months after the second vaccination. In addition, the proportion of patients for whom the vaccine provided little or very limited, if any, symptomatic relief from viral infection was significantly higher in the AIH group. Contrastingly, no cases of serious adverse events associated with vaccination, including severe liver injury, were observed in the study population, which confirms the safety and efficacy of vaccination. Since the battle between humans and COVID-19 will probably continue for a long time, continued and widespread vaccination, particularly for patients for whom medical immunomodulation is essential, is desirable.


There is an urgent need for novel biomarkers to improve the early diagnosis of rheumatoid arthritis (ERA). Current serum biomarkers used in the management of ERA, including rheumatoid factor and anti-cyclic citrullinated peptide (ACPA), show limited specificity and sensitivity. Here, we used metabolomics to uncover new serum biomarkers of ERA.We applied an untargeted metabolomics approach including gas chromatography time-of-flight mass spectrometry in serum samples from an ERA cohort (n=32) and healthy controls (n=19). Metabolite set enrichment analysis was performed to explore potentially important biological pathways. Partial least squares discriminant analysis and variable importance in projection analysis were performed to construct an ERA biomarker panel.Significant differences in the content of 11/81 serum metabolites were identified in patients with ERA. Receiver operating characteristic (ROC) analysis showed that a panel of only three metabolites  could correctly classify 96.7% of patients with ERA, with an area under the ROC curve of 0.963 and with 94.4% specificity and 93.5% sensitivity, outperforming ACPA-based diagnosis by 2.9% and, thus, improving the preclinical detection of ERA. Aminoacyl-tRNA biosynthesis and serine, glycine, and phenylalanine metabolism were the most significant dysregulated pathways in patients with ERA.A metabolomics serum-based biomarker panel composed of glyceric acid, lactic acid, and 3-hydroxisovaleric acid offers potential for the early clinical diagnosis of RA. Given tMetabolomics is a powerful technology to explore changes in the abundance of metabolites within a biological system and can provide valuable insights into the metabolic pathways that are perturbed in different diseases, including RA .Metabolite levels in the serum and urine of patients with RA can exhibit significant changes compared with healthy individuals or individuals with other conditions. These changes are typically associated with the underlying inflammatory processes, immune dysregulation, and oxidative stress commonly observed in patients with RA . AlthougDysregulation of lipid metabolism is also a common trait in RA, and elevated levels of cholesterol and triglycerides have been reported in the serum of patients . Indeed,Alterations in metabolite levels have also been found to reflect different therapeutic effects in patients with early RA (ERA). Metabolites such as uric acid, taurine, methionine, glycine, histidine, and hypoxanthine have been found to be elevated in the serum of patients with ERA after responding to methotrexate treatment with respect to patients for whom the drug was not effective . Contrasversus a matched control cohort to identify a metabolic-related panel to detect ERA. Putative signaling pathways related to dysregulated metabolites were also analyzed to better understand the onset of the disease.Monitoring metabolic changes in ERA may aid in understanding disease progression and treatment response. Although several biofluid metabolomic biomarkers have been reported as useful in the stratification of patient-response prediction to a specific therapy, very few studies have investigated novel biomarkers to improve the early diagnosis of RA. Accordingly, in the present study, we performed serum metabolite profiling in a well-characterized and representative cohort of patients with ERA naïve to treatment 22.1This is an observational case-control study. Patients were recruited at the Joan XXIII University Hospital in Tarragona (Spain). Patients (n=32) were classified as having ERA according to ACR/EULAR 2010 criteria, with a symptoms duration of ≤6 months and not receiving treatment with glucocorticoids or synthetic or biological disease-modifying drugs. Control subjects (n=19) were individuals with no relevant medical history. Groups were matched for sex and age. The study was performed according to the provisions of the Declaration of Helsinki, was approved by the local ethics committee, and adhered to current legal regulations . All methods were approved by the Ethical Committee for Clinical Research (CEIM) of the Pere Virgili Research Institute (Ref. CEIM: 047/2021). All participants gave written informed consent. The following exclusion criteria were applied: patients unwilling or unable to provide informed consent; patients having a diagnosis of any systemic inflammatory condition other than RA, such as (but not limited to) juvenile chronic arthritis, spondyloarthropathy, Crohn’s disease, ulcerative colitis, psoriatic arthritis, active vasculitis, or gout (participants with secondary Sjogren’s syndrome were not excluded); history or presence of cardiovascular, respiratory, hepatic, gastrointestinal, endocrine, hematological, neurological, or neuropsychiatric disorders; or any other serious and/or unstable illness that, in the opinion of the investigator, could constitute a risk when taking investigational samples or could interfere with the interpretation of data.2.2Fasted blood was extracted, and glucose, cholesterol, triglyceride, high-density lipoprotein cholesterol, and hepatic and renal profiling were performed using the standard methods. C-reactive protein (CRP), rheumatoid factor, and anti-cyclic citrullinated peptide (ACPA) levels were determined using standard enzymatic methods.2.3The metabolomic analysis was performed at the Center for Omic Sciences . Samples (100 μL serum) were aliquoted into 1.5 mL tubes and mixed with 400 μL of a solution of methanol: water (8:2) containing internal standards. Samples were vortexed and centrifuged for 5 min at 15000 rpm and at 4°C. Supernatants (200 μL) were transferred to new tubes and evaporated in a SpeedVac at 45°C. Samples were then reconstituted with 30 μL of methoxyamine and incubated for 90 min at 37°C, after which time they were silylated with the addition of 45 μL of MSTFA + 1% TMCS at room temperature for 60 min.Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) separation was performed with helium (purity >99.999%) as a carrier gas at a constant flow of 1.1 mL/min. The initial GC oven temperature was 60°C; 1 min after injection, the GC oven temperature was increased by 10°C/min to 320°C and held for 10 min at 320°C. The samples were injected with split mode 1:20 at an injection temperature of 250°C. Detection was achieved using MS in electron ionization (70 eV) mode and full-scan monitoring mode (m/z 50–600) with an acquisition rate of 5 spectra/s. The temperature of the ion source was set at 250°C with the quadrupole at 200°C.Organic acids were identified and semi-quantified using their pure analytical standards. Specifically, ions were selected and used for quantification based on their impact electron spectra (70 eV) and the main specific ions described in the recorded spectra library Fiehn (Agilent-Technologies Inc. USA).2.4https://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie) (The sample size was estimated using G*Power 3.1.9.7 (hologie) . A 2-folAnalysis of the differences between the qualitative clinical data between study groups was carried out using the Chi-square test. The Shapiro-Wilk test was used to assess the normality of clinical, anthropometric, and metabolomics variables. The non-parametric Mann-Whitney U test was used to analyze differences in the anthropometric, analytical data and metabolomic data between controls and patients with ERA. Data were expressed as median . To identify metabolites that could discriminate between controls and ERA, partial least square regression discriminant analysis (PLS-DA) was used. Variable importance projection (VIP) scores were examined to select the best-discriminating variables, whereby a VIP score of >1 was considered important in the discrimination. Binary logistic regressions and receiver operating characteristic (ROC) curve analysis were performed to identify the best discriminating metabolic model between the groups.https://www.metaboanalyst.ca), a package based on R software environment , was used to obtain a better understanding of which metabolic pathways were enriched and were associated with dysregulated metabolites in the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolite library.MetaboAnalyst 5.0 software  was used to perform all the statistical analyses, and GraphPad Prism v9  was used to generate box plots. A p-value of ≤ 0.05 was considered significant in all analyses.33.1We performed untargeted metabolomic profiling of serum from 32 patients with ERA and 19 healthy controls. The baseline characteristics of the participants are described in 3.2To identify the most relevant metabolites that would allow us to correctly identify ERA based on the serum metabolomic profile, we performed a PLS-DA data analysis using data sets of the aforementioned 15 deregulated metabolites. The score plot of the PLS-DA model showed a partial separation between patients and controls . A heat We next performed a VIP analysis to examine the contribution of the 15 metabolites in determining the presence of ERA. We found that 5/15 metabolites had a VIP score ≥1  and were considered important in the model for determining ERA onset  was then back-evaluated with PLS-DA to test the strength of the model. The results showed that subjects were clearly segregated into two differentiated groups: control and ERA .All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. SR-M, AA-C and MRC. had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design: SR-M, AA-C, RF-G and MRC. Acquisition of data: SC-O, MJP-E, NdC-P. Analysis of the data: MM-H, VA-G and interpretation of data. AA-C, SR-M, RF-G and MRC.


The unique “sandwich” structure of the working electrode (WE) printed with this ink enhances its electrical conductivity, leading to high sensitivity, selectivity, and stability, with almost no water film generated between the WE and the ion-selective membrane (ISM), strong ion selectivity, long-term stability, and anti-interference. The lower detection limit of the sensor for Na+ is 0.16 mM with a slope of 75.72 mV per decade. To validate the sensor's usability, we analyzed three sweat samples collected during physical activity, with Na+ concentrations within the typical range for human sweat .In recent years, the integration of flexible printed electronics and electrochemical sensors has emerged as a new approach for developing wearable biochemical detecting devices. Among the materials utilized in flexible printed electronics, carbon-based conductive inks are considered to be crucial. In this study, we propose a cost-effective, highly conductive, and environmentally friendly ink formulation utilizing graphite and carbon black (CB) as conductive fillers, resulting in a very low sheet resistance of 15.99 Ω sq A highly conductive ink printing sensor for sweat detection. This method provides the possibility of inventing a new type of potential sensor with minimal maintenance cost and good stability.Ion-selective sensors (ISS) play a crucial role in advancing analytical chemistry and are the driving force behind the creation of electrochemical sensors, which can detect various sample solutions non-destructively without requiring external energy to neutralize ion activity. Due to their low production cost, ease of operation, sensitive response, and rapid response,11,12 The use of a large capacitor can increase the contact area between the solid-state conductive layer and the ISM, stabilize the interface potential, and improve electron transfer efficiency. A double-layer capacitive sensor can be described as an asymmetric electronic capacitor. One side is the charge carried by the target cation or anion passing through the ISM, and the other side is the electronic charge formed by the electrons or holes in the conductive layer. This type of conductive layer material mainly consists of carbon nanomaterials and metal nanoparticles. The magnitude of the potential depends on the total amount of charge in the double layer.13,14 ASS-ISS prepared from a conductive material with a large redox capacitor is called a pseudo-capacitive type solid-state ion selective sensor. This type of material has both electronic conductivity and excellent ion conductivity due to ion doping. Its response mechanism is to transfer charge from ions to electrons through an oxidation–reduction reaction, then through the conductive substrate, and finally obtain the ion concentration by displaying the voltage.15 Nevertheless, the water layer and the charge-transfer resistance between the solid-state conductive layer and the ISM prevents ASS-ISS from being used on a large scale.Currently, the sensing mechanisms of ASS-ISS mainly include double-layer capacitance and pseudo-capacitance types. The sensing mechanism of a double-layer capacitive sensor is to form a double-layer capacitor similar to an asymmetric capacitor between the solid-state conductive layer and the ion-selective film (ISM).16 Conductive coatings made from traditional carbon materials like graphite and CB, as well as newer carbon materials like graphene and carbon nanotubes, offer excellent electrical conductivity, corrosion resistance, and environmental friendliness, among other benefits, for a variety of printed electronics applications such as batteries and supercapacitors, electrochemical sensors, printed circuit board resistors, printed heaters, and solar energy harvesting.17–22 Screen printing is the primary ink processing method for these applications, and it has attracted widespread attention in the field of flexible electronics due to its low cost, ease of mass manufacturing, and powerful design.23,24 Several types of publications have reported on the application of screen-printing technology to electrochemical devices. V. Mazzaracchio et al.25 developed a wireless flexible device for monitoring sweat pH by electroplating an iridium oxide thin film over a graphite WE, which provided a pH-sensitive layer and measured the pH in the solution. The slope of the response was −0.079 ± 0.002 V per decade. The same research group also introduced a novel type of screen-printed electrode,1 in which the WE was modified by drop-casting a combination of nanomaterial CB and a sodium ISM. The electrode has a detection range of 10−4 M to 1 M for Na+ and a response slope of 58 ± 3 mV per decade. A. Hauke et al.26 created a screen-printed, low-cost chloride ion sensor for remote monitoring of cystic fibrosis (CF) patients with high sensitivity and a detection limit of 2.7 × 10−5 M, covering the entire concentration range for CF diagnostics and available as a portable device at a greatly reduced cost. Furthermore, several literature publications27–29 have demonstrated that screen printing technology can manufacture electrochemical sensors with cheap cost, mobility, large-scale manufacturing, and outstanding performance, which can be used in biosensing, environmental detection, and analysis.Conductive ink is one of the most crucial materials in the field of printed electronics.30–32 Additionally, the physical and chemical properties of carbon materials facilitate the stable passage of electrodes. Building upon this understanding, we have developed a novel method for manufacturing SPE capable of detecting Na+ concentrations. Our approach employs conductive ink comprising graphite and CB, and screen-printing technology to fabricate carbon electrodes. Based on the foregoing, we disclosed a new technique for manufacturing a screen-printed electrode for detecting Na+ concentration, conductive ink made from graphite and CB, and a carbon electrode made using screen printing technology .y Fig. S1. It has + content of around 40 mM. We reported an All-solid-state sodium ion selective sensor (ASS-Na-ISS) in a NaCl concentration gradient solution. The linear fitting slope is 75.72 mV per decade when evaluating the potential difference between the ASS-Na-ISS and the reference electrode (RE). As far as we know, this is the first time a superior reaction has been found in earlier literature studies. Additionally, the electrode offers advantages such as long-term stability, excellent printing performance, low cost, and the ability for large-scale production. The performance of the electrode was evaluated in both Na+ solution and sweat, demonstrating its high potential for use in biosensing and detection. These findings broaden the scope of future applications for electrochemical flexible devices.The potential difference, according to the Nernst equation, is linear with the logarithm of the ion concentration to be measured. Human perspiration has a typical NaCB was purchased from Cabot Corporation. Graphite was obtained from Qingdao Jintao Graphite Co., Ltd. Defoamer (BYK-022) was purchased from BYK Chemistry. Dispersant (T-859) and waterborne polyurethane resin were purchased from Shenzhen Jitian Chemical Co., Ltd. PEDOT/PSS was obtained from 3AMaterial. Sodium ionophore III (ETH2120) was purchased from Shanghai Yuanye Biotechnology Co., Ltd (MedMol). Potassium tetrakis-(4-chlorophenyl)-borate (KTClPB) and bis-(2-ethylhexyl)-sebacate (DOS) was purchased from Aladdin. Polyvinyl chloride (PVC), cyclohexanone (CHA), and TritonX-100 were got from Shanghai Macklin Biochemical Co., Ltd. Ag/AgCl paste was purchased from Shanghai Julong Electronic Technology Co., Ltd. All chemicals and solvents were used as received without further treatment.Table S1To explore the influence of carbon content on electrical conductivity, the carbon content was set as 20%, 25%, 28%, 30%, 32%, and 35% (w/w) in six groups , the quality of other components remained unchanged.The printing plate pattern is designed, as shown in Fig. S2.Take 1 mL PEDOT/PSS and mix it with 1‰ (V/V) TritonX-100. Leave the mixture at 4 °C for 4 h to ensure it is well mixed. Apply the mixture to the centre area of the WE of the printed electrode using a pipette gun. Allow the mixture to freely diffuse, and once it has covered the entire surface of the WE, place it in an oven and dry it at 40 °C to create a solid electrolyte layer.+-ISM. To prevent interference, apply insulating material to all exposed areas and save the electrodes.Sodium ionophore III (1% w/w), ion exchanger , high polymer , and plasticizer  combined with CHA (33 mg PVC per 350 μL CHA). Sonicate it for 10 minutes to ensure that it is evenly mixed, then let it aside for 1 hour before applying it onto the solid electrolyte layer with a pipettor. When the membrane liquid has completely covered the whole solid dielectric layer, dry the electrode in an oven at 40 °C to make a NaSuzhou Jingge Electronics Co., Ltd's four-probe tester is used to test the conductivity (sheet resistance) of the ink. All electrochemical performance tests are carried out on the Autolab PGSTAT 302N produced by Metrohm. Field emission scanning electron microscopy  and transmission electron microscopy  were used to observe the microstructure. Raman spectroscopy  was used to characterize the internal structure.34 also can reducing the production cost. CB is a nano-scale conductive ball with a larger specific surface area than graphite. While graphite has high lateral conductivity on graphite sheets, its longitudinal conductivity between graphite sheets is substantially lower. In contrast, minute CB particles are more likely to scatter across graphite gaps and act as a bridge,35 generating a “sandwich” structure. So these two materials are more conductive when combined because the gaps between the conductive particles in this “sandwich” structure are smaller than either one alone. As a result, the conductive ink formed by combining these two materials has strong conductivity, which can effectively increase the transmission efficiency of the WE. This can lead to improved sensitivity and reaction time performance of the sensor. Additionally, carbon nanoparticles have a high specific surface area and are hydrophobic, which has been shown to improve potential drift, anhydrous layer interference, and the potential stability of electrochemical sensors.36Graphite is a layered material, and ball milling can thin the graphite layers to form few-layer graphite (a graphene-like structure). This can increase the conductivity of graphite and make it more similar to graphene,37 As the ball milling duration exceeds 4 h, the sheet resistance of the ink essentially remains unchanged. This may be due to that the ball milling process has reached its saturation point and the reduction in the contact resistance. Thus, the sheet resistance remains constant. The lowest sheet resistance was observed on the G/C-30 printed electrode after ball milling for 4 h, which is 15.99 Ω sq−1 (electrode thickness is 25 μm). According to the relationship between sheet resistance and conductivity:In composite conductive inks, graphite and CB are used as conductive fillers, and their content has the greatest direct impact on the ink's conductivity. 3 S m−1. σ is conductivity, ρ is resistivity, Rs is sheet resistance and d is the thickness of the electrode.The conductivity is calculated as 2.5 × 10To summarize, it can be concluded that G/C-32 and G/C-35 inks are not ideal for printing due to the formation of agglomerates and uneven surfaces resulting from the oversaturation of carbon content, which can adversely affect performance. Therefore, further study on these inks will be avoided. On the other hand, G/C-30 ink showed the highest conductivity among the experimental groups and was chosen for the construction of ISS.38,39 This conclusion also is demonstrated via Raman spectrum analysis. The Raman spectra of the conductive ink before and after ball milling is shown in I2D/IG increased by 0.185. It has been demonstrated that the ball milling technique mechanically exfoliates graphite during the creation of conductive inks, resulting in the few-layer graphene.40 The characterization results indicate that ball milling can effectively disperse the CB particles in the ink, resulting in an even distribution among the graphite flakes and the formation of a “sandwich” structure. This leads to a reduction in contact resistance between the graphite flakes and an increase in conductivity between them. Additionally, ball milling can increase the number of graphite layers, resulting in the generation of few-layer graphene, which significantly improves the ink's conductivity.41,42 This is consistent with previous studies that have shown that electrodes modified with nano CB have lower background currents than those modified with carbon nanotubes42,43 and graphene oxide,44 thus improving sensor measurement accuracy. Furthermore, the composite ink used in this study for printing electrodes has a lower cost compared to materials such as carbon nanotubes and graphene, making it more suitable for large-scale industrial production.Then, the cyclic voltammetry test was done on the screen-printed three-electrode system. We used G/C-20, G/C-25, G/C-28, and G/C-30 ink-printed electrodes, as well as pure graphite-printed (GI) electrodes, in the potential range of −1 V to 1 V in 0.1 M NaCl solution. Impedance experiments were conducted on several electrodes across the frequency range of 0.1 Hz to 100 kHz, as illustrated in −4 to 1 M, we investigated the ASS-Na-ISS's detecting potential. According to the Nernst equation, the electromotive force (EMF) has a linear relationship with the logarithm of the ion concentration to be measured. + concentration in human sweat (about 40 mM). The ASS-Na-ISS was further calibrated in the range of Na+ concentration from 10 mM to 70 mM, as shown in + concentration of human sweat, excluding spots outside the detection limit. In Fig. S4,We started studying the performance of the ASS-Na-ISS once it was built. In a concentration gradient NaCl solution of 10The repeatability was then assessed using three samples from the same batch . After c+ concentrations of 51 ± 4 mM, 39 ± 5 mM, and 46 ± 2 mM. The steep slope observed in the report is rare, indicating a high sensitivity of the ASS-Na-ISS. To evaluate the usability of the ASS-Na-ISS for practical applications, three sweat samples were collected from participants during exercise, with NaAside from sensitivity and detection limit, the ASS-Na-ISS's stability, selectivity, reversibility, and water layer test are all key techniques to evaluate its performance.2, 5 mmol KCl, 5 mmol MgCl2 and 5 mmol HCl. As shown in 2 mixed solution, 0.1 M NaCl and 0.1 M MgCl2 mixed solution, and 0.1 M NaCl and 0.1 M HCl mixed solution. As indicated in + and other exogenous cations and organic substrates do not affect electrode sensitivity.The selectivity test for ASS-Na-ISS is performed in an electrochemical reaction cell with rotor stirring, and the potential change is measured by successively introducing the ions under examination. Initially, the test response cell only contains 100 mL of deionized water. The ions are added in the following order: 5 mmol CaCl−1. Table S21,45–50 When compared to previous work, the drift potential of the ASS-Na-ISS developed in this study falls within the range reported in these publications. In addition, the ASS-Na-ISS's storage stability was studied. Its storage stability was evaluated by measuring the induced potential on the day of production as well as after 5, 10, and 15 days in 0.1 M NaCl solution, as shown in To assess the stability of the ASS-Na-ISS over time, the potential is measured continuously for 24 h in a 0.1 M NaCl solution to verify its operational stability. As shown in The ASS-Na-ISS's photosensitivity was further examined by exposing it to indoor light, darkness, and UV light and measuring the potential response in 0.1 M NaCl solution. During alternating light source illumination, there was no discernible potential shift .3 solutions and KOH. The pH sensitivity of the sensor is shown in The pH effect of the ASS-Na-ISS was tested over the range of 2–12 for 0.1 M NaCl solutions and 0.01 M NaCl solutions. The pH was adjusted with HNO+ concentrations.The reversibility of the ASS-Na-ISS was also investigated by varying NaCl concentrations between 0.1 M and 0.01 M. As depicted in 1,51,52 To determine whether a water film exists, immerse the electrode in a 0.1 M NaCl solution. After 30 min of steady potential, remove the ASS-Na-ISS and immerse it in a 0.1 M KCl solution. Return the potential to the ASS-Na-ISS when it has stabilized for 30 min. In the original NaCl solution, record the time-dependent change curve of the induced potential, as illustrated in The ASS-Na-ISS was also subjected to a water layer test. The presence of a water film between the ISM and the electrode contributes significantly to the measurement instability of the ASS-ISS.The remarkable electrochemical properties, stability, and cost-effectiveness of carbon nanomaterials have had a significant impact on the advancement of potentiometric sensors. In this study, we have developed a screen-printed sensor for detecting sweat. By harnessing the unique properties of graphite and CB, we have developed highly conductive and eco-friendly inks. The use of this ink in printing the WE lead to sensors that do not form water film, and compared to some of the studies in Table S2,All authors contributed to the study's conception and design. Material preparation was performed by Wanggang Zhang and Jian Wang. Data collection and analysis and the first draft of the manuscript were performed by Dengke Wang. Review and editing were performed by Xiaohong Li. Yiming Liu provided financial support for this project. All authors read and approved the final manuscript.There are no conflicts to declare.RA-013-D3RA01410J-s001


Automatic chest compression devices (ACCDs) can promote high-quality cardiopulmonary resuscitation (CPR) and are widely used worldwide. Early application of automated external defibrillators (AEDs) along with high-quality CPR is crucial for favorable outcomes in patients with cardiac arrest. Here, we developed an automated CPR (A-CPR) apparatus that combines ACCD and AED and evaluated its performance in a pilot animal-based study.Eleven pigs ] group) were enrolled in this study. After 2 min observation without any treatment following ventricular fibrillation induction, CPR with a 30:2 compression/ventilation ratio was performed for 6 min, mimicking basic life support (BLS). A-CPR or C-CPR was applied immediately after BLS, and resuscitation including chest compression and defibrillation, was performed following a voice prompt from the A-CPR device or AED. Hemodynamic parameters, including aortic pressure, right atrial pressure, coronary perfusion pressure, carotid blood flow, and end-tidal carbon dioxide, were monitored during resuscitation. Time variables, including time to start rhythm analysis, time to charge, time to defibrillate, and time to subsequent chest compression, were also measured.There were no differences in baseline characteristics, except for arterial carbon dioxide pressure , between the two groups. There were no differences in hemodynamic parameters between the groups. However, time to charge , time to defibrillate , and time to subsequent chest compression  were shorter in the A-CPR group than in the C-CPR group .A-CPR can provide effective chest compressions and defibrillation, thereby shortening the time required for defibrillation. Cardiopulmonary resuscitation (CPR) and defibrillation are important for survival of patients with sudden cardiac arrest  and an eFig 1). The functional part is mounted on the supporting frame, which is composed of a compression-performing apparatus, an AED, and a control panel. The compression-performing apparatus is composed of a piston and an actuator and is used to compress the chest. The piston is a round bar connected to the actuator operated by a battery. The compression depth and rate of compression range from 0 to 6 cm and 0 to 120 compressions per minute, respectively, and can be adjusted. An AED is integrated with the compression-performing apparatus. The defibrillation electrodes are stored in the designated pocket. The control panel controls chest compression and automated defibrillation. The operator can control the start or stop of chest compression, compression depth, compression rate, and shock delivery. The support frame consists of a backboard and a supporting structure on which the compression-performing apparatus and AED are mounted. The sequence of chest compression and defibrillation is based on the basic life support (BLS) algorithm and is installed into the A-CPR [Fig 2).A-CPR consists of a functional part and a supporting frame  performed using an ACCD and an AED in a swine model of cardiac arrest. This study was approved by the Institutional Animal Care and Use Committee of Yonsei University Wonju College of Medicine, Wonju, Republic of Korea (YWC-210517-1).2) (35–45 mmHg). Under aseptic conditions, the right femoral artery was cannulated, and aortic blood pressure was continuously recorded using a 5-F micromanometer-tipped catheter. Right atrial pressure was recorded using a 5-F micromanometer-tipped catheter inserted through the right external jugular vein. A vascular flowmeter  was placed on the right internal carotid artery to monitor carotid blood flow (CBF). A 5-F pacing catheter was introduced through the right internal jugular vein to induce VF. Once the catheters were in place, a 1,000 unit intravenous (IV) heparin bolus was administered to prevent thrombosis, and baseline arterial blood gas analyses were performed using a blood gas analyzer .Twelve Yorkshire pigs  were used in this study. The pigs were allowed full access to water and food until the day before the experiment and were fasted from midnight. After sedating the animals with intramuscular ketamine (15 mg/kg) and xylazine (2 mg/kg) followed by inhalation of 3% isoflurane, endotracheal intubation was performed with a cuffed endotracheal tube. The animals were ventilated with oxygen and nitrous oxide via a volume-controlled ventilator  with a tidal volume of 10 mL/kg and a ventilation rate of 18 breaths/min adjusted to maintain normal arterial oxygen saturation (94–98%) and end-tidal carbon dioxide  and pads were also applied before the start of the experiment to reduce the bias from any delay in applying AED pads.The pigs were randomized into two groups using randomization envelopes containing different CPR methods (A-CPR or C-CPR group). An ACCD or A-CPR was positioned before the experiment to minimize the risk of catheter or the monitoring apparatus dislodging. AED .After the baseline data measurement, VF was induced by delivering an alternating electrical current of 60 Hz to the endocardium, which was confirmed by the electrocardiogram (ECG) waveform and a decrease in aortic pressure. After 2 minutes of untreated VF, basic BLS was performed for 6 minutes to mimic a BLS situation in which a bystander recognizes cardiac arrest and calls for help. Mechanical chest compressions with a depth of 5 cm using an ACCD  or A-CPR were performed at a rate of 100 chest compressions/min and a compression/ventilation ratio of 30:2 was maintained. Positive pressure ventilation with a room air at a tidal volume of approximately 300 mL was delivered using a resuscitator bag . After 6 min of BLS, defibrillation was performed according to the instructions (voice prompt) from the AED for the C-CPR group and the A-CPR device for the A-CPR group. After defibrillation, chest compression administered continuously while single ventilation with 100% oxygen were performed every 6 s, mimicking advanced life support (ALS) (The experiment was terminated immediately after 2 min of ALS. Once a pig achieved return of spontaneous circulation (ROSC), the animal was euthanized by IV potassium chloride injection.2, and CBF were continuously recorded. Coronary perfusion pressure (CPP) was calculated as the difference between the aortic and right atrial pressures in the end-diastolic phase. Outcome measures included chest compression fraction (CCF), rate of successful defibrillation, and ROSC. Time variables, including time to start rhythm analysis, time to charge, time to defibrillation, and time to subsequent chest compression, were measured.Data were digitized using a digital recording system . Aortic and RAP, ETCOt-test or the Mann–Whitney U test was used to compare the continuous variables between the A-CPR and C-CPR groups, as appropriate. The nominal variables are reported as counts and percentages and were compared using the chi-square or Fisher exact test, as appropriate. A linear mixed-model analysis was used to compare hemodynamic parameters, including aortic systolic pressure, aortic diastolic pressure, mean RAP, CBF, CPP, and ETCO2 between the two groups. The statistical results are presented as group-time interaction. Results with P < 0.05 were considered statistically significant. Data were analyzed using the Statistical Package for the Social Sciences Statistics version 23.0 for Windows .Continuous variables are presented as mean ± standard deviation or medians and interquartile ranges according to the normality of distribution. Student’s 2, in baseline characteristics between the groups (Table 1).Initially, six pigs were enrolled in each group; however, one in the A-CPR group was excluded because of AED malfunction. Thus, five pigs in the A-CPR group and six pigs in the C-CPR group were included in the final analysis. There were no significant differences, except PaCOFig 4).There were no significant differences between the groups in the group-time interaction analyses of hemodynamic variables . There were no differences in ROSC and successful defibrillation because spontaneous circulation was restored in all animals after the first defibrillation and after 2 min of ALS.Table 2).Time to charge (28.9 ± 5.6 s in the A-CPR group vs. 47.2 ± 12.4 s in the C-CPR group), time to defibrillation (29.1 ± 7.2 s in the A-CPR group vs. 50.5 ± 12.3 s in the C-CPR group), and time to subsequent chest compression (32.4 ± 6.3 s in the A-CPR group vs. 56.3 ± 10.7 s in the C-CPR group) were shorter in the A-CPR group than in the C-CPR group  Click here for additional data file.


To evaluate whether the volume of wash out rinse after povidone iodine (PI) application for intravitreal injections (IVI) affects patients’ ocular surface irritation.This was a prospective, single-masked, randomized-controlled trial consisting of 142 subjects. A total of 51, 45, and 46 patients received 3-mL, 10-mL, and 15-mL of ocular rinse respectively. Reductions in the Ocular Surface Disease Index (OSDI) and the Standardized Patient Evaluation of Eye Dryness II (SPEED II) surveys, conducted before and at 24–72 h post-injection, were analyzed.p-value = 0.788), tear break-up time , Oxford fluorescein grade  between the study groups prior to injections. Dry eye symptoms as measured by reductions in the OSDI and SPEEDII scores were not different between the study groups .There was no statistical difference in objective dry eye findings of Schirmer test (There is no difference in patients’ ocular surface irritation between 3-mL, 10-mL, and 15-mL post injection rinse. Given the large number of IVIs performed, modification of practice patterns based on these findings could lead to significant reduction in global cost burden for IVIs.The online version contains supplementary material available at 10.1186/s40942-023-00470-z. Intravitreal injections (IVI) are the most commonly performed ophthalmic procedures in the United States . 5% PoviPrevious guidelines have emphasized anti-septic techniques before and analgesia during injections . HoweverInterestingly, while rinsing with a buffered solution after IVI is common practice , to our This study was a single-center, prospective, randomized-controlled, single-masked clinical trial conducted at the Dean McGee Eye Institute, Department of Ophthalmology, University of Oklahoma Health and Sciences Center, Oklahoma City, OK. USA. Ethics approval was obtained from the university’s Institutional Review Board (IRB) (IRB #9810) with adherence to the tenets of the Declaration of Helsinki. All study participants were provided with written informed consent.Patients requiring IVI for their clinical condition during the study period (March-July 2021) were identified as potential participants. Exclusion criteria included active ocular infection, eyelid trauma, graft versus host disease, thyroid eye disease, pregnancy, and lack of the ability or willingness to participate in the study, which included answering the post-injection telephone questionnaire 24–72 h after IVI.Patients were masked and randomized to 3-mL, 10-mL, and 15-mL washout cohorts in a 1:1:1 ratio. A computer-based random number-generating algorithm was used for randomization. One eye per patient was evaluated in cases of bilateral injections. The cohort distribution is displayed in Fig. Power analysis: Our preliminary data showed that the mean (SD) OSDI score reduction is 1.6 (22.1), 7.7 (12.7), and 13.8 (15.6) for the 3 ml, 10 ml, and 15 ml treatment group, respectively. To detect the difference between the 3 ml and the 15 ml group with the observed SDs and 0.05 alpha level (two-sided), 40 patients per group were needed to achieve 80% power based on a two-sample t-test with unequal variance. Our preliminary data also showed that the mean (SD) SPEED II score reduction is 1.8 (4.4), 1.4 (6.6), and 5.1 (3.9) for the 3 ml, 10 ml, and 15 ml treatment group, respectively. With 40 patients per group, we had 94% power to detect the difference in SPEED II score reduction between the 3 ml and the 15 ml group, again based on a two-sample t-test with unequal variance with 0.05 alpha level (two-sided). To account for 20% drop-out rate, an enrollment of 50 patients per group was planned. The PASS 16 software was used for the power analyses.p-values were considered statistically significant when they were < 0.05.Statistical analysis: Descriptive statistics were used to summarize basic demographic characteristics. Continuous data were summarized using mean and standard deviation. Comparisons between treatment groups were performed using the Chi-square test for categorical variables and analysis of variance (ANOVA) for continuous variables. Multiple comparisons were adjusted by Tukey’s method. The OSDI score was calculated via the formula: OSDI = [ × 100] / [ × 4] . The tot160 patients enrolled in the study. One patient subsequently met the exclusion criteria and was not randomized. 17 dropped out of the study giving a total of 142 patients for the follow-up and for the final analysis  or the SPEEDII score   vs. larger washout (10 or 15-mL) volumes using two different standardized ocular surface irritation questionnaires (OSDI and SPEEDII). It is possible that 3-mL of rinse with purified water solution may be sufficient to remove all residual PI or that the difference in residual PI in 3-mL vs. 10 or 15-mL of washout is not symptomatically significant. However, there was a trend of overall reduction in surface irritation symptoms after washouts in all 3 cohorts Fig. . This suAdditionally, we repeated the questionnaires within 72 h of IVIs. Both OSDI and SPEEDII assess symptoms within the past several days and hence, theoretically, differences in symptoms from the time of injection to the time of the phone survey should have been captured. However, recall bias towards symptoms at the time of the phone survey may overshadow discomfort immediately after IVIs. Future studies with surveys conducted at a shorter time interval, e.g. less than 24 h, may elucidate a difference between various washout volumes. However, our study shows that by 1–3 days after IVI, patients do not report a significant difference in surface irritation symptoms. A confounding factor could be number of injections previously received by patients. Almost all of our patients had received multiple prior injections; however, we did not analyze whether individual cohorts had a statistically significant difference in number of prior injections. We did show that the cohorts did not have significant difference in race, gender, and pre-existing dry eye disease or symptoms.While on an individual scale, the difference of 12-mL of rinse volume may not seem significant, globally it has a significant cost and time burden. Ocular rinses are inexpensive and cost is approximately 3 cents per mL . However, by 2016, 5.9 million IVIs were being performed in the USA and this number continues to increase exponentially . This woThe strength of this study includes a randomized controlled design with variation of a single variable, the rinse volume. This, together with the single masked design, eliminates ascertainment biases. Additionally, the patient’s lack of knowledge of which treatment they received reduces the possibility of placebo effects when completing questionnaires. The number of participants were sufficient to ensure lack of statistical significance in unwanted variables such as gender, eye of study, and pre-treatment objective dry eye findings. Limitations of the study include lack of post-treatment objective measures as well as single time-point evaluation after the washout. It is possible, that objective findings, while not the goals of the study, can be affected by rinse out volumes. Furthermore, we did not stratify patients into ocular surface disease severity  subgroups within the three study groups. We attempted to mitigate this potential confounding factor by performing objective and subjective assessment of patients prior to IVI; we did not note any statistically significant differences between the three groups Table . Future In conclusion, we noted that different volumes of ocular rinse solution did not affect patients’ experience of ocular surface irritation after IVIs. Ultimately, the decision to use a certain volume of ocular rinse should be based on clinicians’ experience and patients’ expectations. A personalized approach with appropriate informed consent should be undertaken whenever possible. Clinicians may consider our findings as they optimize IVI protocols to improve efficiency and economy.Below is the link to the electronic supplementary material.Supplementary Material 1


The GWAS using the legacy data detected several known agriculturally important genes, indicating reliability of the legacy data for GWAS. By comparing the GWAS using legacy data (L-GWAS) and a GWAS using phenotypic data that we measured (M-GWAS), we detected reliable QTL for agronomically important traits. These results suggest that an L-GWAS is a strong alternative to replicate tests to confirm the reproducibility of QTL detected by an M-GWAS. In addition, because legacy data have often been accumulated for many traits, it is possible to evaluate the pleiotropic effect of the QTL identified for the specific trait that we focused on with respect to various other traits. This study demonstrates the effectiveness of using legacy data for GWASs and proposes the use of legacy data to accelerate genomic breeding.Genome-wide association studies (GWASs) are used to detect quantitative trait loci (QTL) using genomic and phenotypic data as inputs. While genomic data are obtained with high throughput and low cost, obtaining phenotypic data requires a large amount of effort and time. In past breeding programs, researchers and breeders have conducted a large number of phenotypic surveys and accumulated results as legacy data. In this study, we conducted a GWAS using phenotypic data of temperate  Crop improvement through genomic breeding is essential to increase crop productivity and to feed the growing global population . Identifhttps://www.gene.affrc.go.jp/distribution-plant_en.php). We performed the GWAS with 198 temperate japonica rice (Oryza sativa L.) varieties and found several known agriculturally important genes. We also conducted a GWAS using phenotypic data that we measured, referred to as a measured-GWAS (M-GWAS), and compared this with the results of the GWAS using legacy data, referred to as a legacy-GWAS (L-GWAS). Several genomic regions were detected in both the M-GWAS and L-GWAS, where previously unknown QTL are probably located. This demonstrated that the combination of an L-GWAS and M-GWAS is effective in detecting reliable QTL. In addition, we evaluated the pleiotropic effects of QTL by comparing GWASs for multiple traits using legacy data. Here, we show the availability of legacy data for accelerating basic research and practical genomic breeding.To evaluate the validity of legacy data, we performed a GWAS on various rice phenotypic data retrieved from a public database provided by the NARO Genebank in Japan , and moderately correlated with Lemma and Palea Color (LPC) (r = 0.4), but not with Brown Rice Color . Plant Type (PT) was highly positively correlated with Panicle Number , while negatively correlated with Culm Length (CL), Panicle Length (PL), Culm Thickness (CT), and Spikelet Density (SD) . This suggests that the PT, which is evaluated by the breeders’ intuition, is the result of the integration of these traits. In this case, by performing a GWAS on PT and the individual traits and comparing the loci controlling these traits, we were able to examine the relationship between PT and these specific traits in detail (see below).To perform GWASs efficiently, a genetically highly structured population is not desirable. A principal component analysis (PCA) was conducted to measure the population structure of 198 temperate e L-GWAS  and 172 e M-GWAS . For bote M-GWAS . A totale M-GWAS . Among te M-GWAS . As showThe distributions of the 33 traits in the legacy data were examined in histograms . Among tEpidermal Patterning Factor-Like protein 8 (EPFL8)/Regulator of Awn Elongation 2 (RAE2)/Grain number, grain length and Awn Development 1 (GAD1) is located (waxy is located (QTL of seed shattering in chromosome 1 (qSH1) is located , Endosperm Type (ET), and Seed Shattering (SS), which we considered to be suitable for evaluating whether the L-GWAS is effective because genes that have a major effect on each trait have been identified. In the GWAS for AP, a strong peak was detected on chromosome (Chr.) 8 ; within  located . A stron located . In the  located , where w located . In the  located , where Q located . These rHEN1 suppressor 1 (HESO1), Heading date 1 (Hd1), and Heading date 2 (Hd2) . For this purpose, we first focused on comparing results from the L-GWAS for Heading Date at Niigata (HDN) and those from the M-GWAS for the Heading Date measured at Nagoya. There were several peaks in the Manhattan plot of the L-GWAS; some of these overlapped with known heading genes, such as  2 (Hd2) , and the 2 (Hd2) . For Hd1d2)  of single-nucleotide polymorphisms (SNPs) between the L-GWAS and M-GWAS within the peak regions (HESO1 is located (10(P) of these polymorphisms was very high  between the L- and M-GWASs . Application of Method 1 to the se peaks . For Met regions . In the  located , there w M-GWASs . In a sise peaks . We alsose peaks . Althougse peaks , the pease peaks  and a hiHd1, HESO1, Hd2, and peak1) were also detected in this L-GWAS . Data of Heading Date measured at different locations are available in legacy data of NARO Genebank and using these data would improve the statistical accuracy of the GWAS by increasing repeatability and decreasing missing values. However, there is also concern that mixing data from different locations could reduce the accuracy of the GWAS, because the influence of a QTL could differ between different locations. With these considerations in mind, we conducted an L-GWAS of the Integrated Heading Date (IHD) using all data recorded at all different locations . All fous L-GWAS . In addis L-GWAS . The ides L-GWAS  and a hiR2 = 0.64; R2 = 0.56; In the same way, we compared GWASs on the amount of White Core (WC) using the legacy data and the data that we measured . BecauseThese case studies show that the combination of L- and M-GWASs is an easy and available method to find QTL for agronomically important traits. In particular, WC is known to be greatly affected by environment, such as temperature during the ripening period , and is OsC1) (OsDFR)  had a non-colored ApC regardless of the haplotype of OsDFR, while the functional OsC1 lines had a colored ApC  for ApC, while OsC1/osdfr showed a light-color (scores 2–4) (OsC1 determines whether the apiculus is colored or non-colored whereas OsDFR regulates the degree of ApC (i.e. color tone). Previous studies have shown that OsC1 acts as a switching gene for regulation of anthocyanin synthesis and activation of the expression of OsDFR and other related genes, and OsDFR is involved in the branching of anthocyanin synthesis pathways  . Second, (OsDFR) . There al allele   . These rres 2–4) , suggestpathways . The rese organs . On the or OsDFR , indicatNALLOW LEAF 1 (NAL1) (10(P) values of CT and SD were relatively low, but the peak disappeared by Method 1  values of CT were relatively low, but the peak disappeared by Method 1  , which c1 (NAL1) . From ou1 (NAL1) . The −loMethod 1 . On the Method 1 . Thus, p–28.8 Mb , PN show–28.8 Mb . CT also–28.8 Mb , but CL,–28.8 Mb . Chigira–28.8 Mb  and comp–28.8 Mb . Peak 2 –28.8 Mb , suggest–28.8 Mb , PN, CL,–28.8 Mb . The −loMethod 1 . On the Method 1 . ConsequMethod 1 . The resMethod 1 . These rUsing rice phenotypic data from the NARO Genebank as an example, this study shows that legacy data are very useful for GWASs. The advantages of using legacy data revealed by this study are as follows. First, the process of acquiring trait data, which requires a lot of time and effort, can be greatly reduced by allowing legacy data to be used in place of newly acquired data, as like a kind of blinded experiment. Also, by comparing the legacy data and our own acquired data, simple mistakes , potato (Solanum tuberosum), grasses, fruit trees, and vegetables. In addition to the NARO Genebank data, there are reports investigating yield traits such as fertility, germination characteristics  panels comprising 198 (for L-GWAS) and 172 (for M-GWAS) varieties, which were collected from various places in Japan  was used to provide information on gene position and coding sequences.SnpEff software version 4.3 T  was usedGWAS in the R package “rrBLUP” version 4.3 with default parameter settings (association.test in the R package “gaston” version 1.5.7 with default parameter settings (Meff) . If a peak detected in the L-GWAS is the same as a peak in the M-GWAS, the peak in the L-GWAS disappears when marker genotype data of the peak in the M-GWAS are included as a fixed effect in the GWAS model because explainability of the peak in the L-GWAS is removed by the fixed effect (10(P) of SNPs within the peak region were calculated (Method 2). When comparing L- and M-GWASs, only SNPs that were present in both L- and M-GWASs were extracted and analyzed for correlation. The effect size of each SNP was calculated using an in-house script that is a modification of the function GWAS in the R package “rrBLUP” version 4.3. To enable direct comparison of SNP effects between the traits, standardized phenotypic values were used for the calculation . Then, the absolute values of the SNP effects were provided for the comparison.The population structure of the 198 and 172 varieties was estimated using PCA performed using the R package “SNPRelate” version 4.2 . For GWAsettings . For binsettings . In boths (Meff) . In thisd effect . In addihttps://www.gene.affrc.go.jp/distribution-plant_en.php). Original phenotypic data were scored according to the in-house manual (available in the NARO Genebank website), with five replications for measurement traits and observing the entire survey area for the observational traits. In the NARO Genebank, there are phenotypic data measured at 12 locations  from 1965 to 2020. The numerical data were used without modification. The Heading Date was converted to numerical data as days after July 1. Ordinal data, such as color traits, were converted to numerical data according to the scale in the NARO Genebank (lmer function of the R package “lme4” version 1.1-31 (Phenotypic data were downloaded from the NARO Genebank (accessed August 5, 2022; Genebank . Basicaln 1.1-31 . For Hean 1.1-31 . The traWC and Heading Date used for M-GWAS were surveyed at Togo Field, Field Science Center, Nagoya University, in 2014 and 2015, respectively. Heading Date is the number of days from planting to heading dates. The survey of BW was conducted at the experimental field of Fukui Prefectural University in 2014. These phenotypic data used for M-GWAS are shown in TukeyHSD function and anova function in R.Multiple comparison tests were performed using the EPFL8/RAE2/GAD1, LOC_Os08g37890; waxy, LOC_Os06g04200; qSH1, LOC_Os01g62920; HESO1, LOC_Os01g62780; Hd1, LOC_Os06g16370; Hd2, LOC_Os07g49460; OsC1, LOC_Os06g10350; OsDFR, LOC_Os01g44260; NAL1, LOC_Os04g52479.Sequence data from this article can be found in the RGAP data libraries under the following accession numbers: kiad018_Supplementary_DataClick here for additional data file.


Supramolecular hydrogels obtained from the self-organization of simple peptides, such as tripeptides, are attractive soft materials. Their viscoelastic properties can be enhanced through the inclusion of carbon nanomaterials (CNMs), although their presence can also hinder self-assembly, thus requiring investigation of the compatibility of CNMs with peptide supramolecular organization. In this work, we compared single-walled carbon nanotubes (SWCNTs) and double-walled carbon nanotubes (DWCNTs) as nanostructured additives for a tripeptide hydrogel, revealing superior performance by the latter. Several spectroscopic techniques, as well as thermogravimetric analyses, microscopy, and rheology data, provide details to elucidate the structure and behavior of nanocomposite hydrogels of this kind. Nanocomposite materials include structures with dimensions in the nanoscale that allow researchers to leverage the exceptional properties of nanomaterials. In recent years, nanocomposites have attracted researchers’ attention in light of their versatile nature, which enables a great variety of applications . The incIn particular, hydrogels are a type of soft matter that benefits from the addition of CNMs to enhance their properties, especially their conductivity, their responsiveness to physicochemical stimuli, and their viscoelastic resistance against applied stresses ,34,35,36The simpler the peptide sequence, the cheaper will be its production. Therefore, di- and tri-peptides stand out as ideal compounds that can also be produced with cheap liquid-phase synthesis on a large scale . Among tThe ability of L-Leu-D-Phe-D-Phe to interact with CNMs to yield hydrogels with enhanced properties was recently investigated. It was found that the addition of oxidized multi-walled CNTs (ox-MWCNTs) yielded hydrogels with the best properties, such as resistance against applied stress relative to graphene oxide (GO) or oxidized carbon nano-horns (ox-CNHs) . Carbon ®, lot no. SP2167,1.2–1.7 nm wide, and 0.1–4 µm long).Reagents and solvents were purchased from Merck  and they were used as supplied, unless indicated differently. All solutions and buffers were prepared using high-purity water, which was obtained from a Milli-Q apparatus  with a resistivity higher than 18.2 MΩ·cm at 25 °C. The sonicator used to disperse the nanomaterials was the Branson Ultrasonic 3800 . SWCNTs were purchased from Nanointegris  according to a published procedure . They we3 (65% w/w), sonicated for 15 min at ambient temperature, and then refluxed for 24 h at 130 °C and 300 rpm. Next, the crude mixture was carefully and slowly poured onto ice-cold water (150 mL) for quenching. The mixture was filtered with a Millipore membrane  and washes were performed with distilled water until neutrality of the filtrate was achieved, then it was washed once with methanol, and once with diethyl ether. The following day the powder was dried in an oven (80 °C).DWCNT oxidation was performed in nitric acid . First, 3 in a flask with a round bottom, and the reaction was placed in a sonicator at 40 °C for 30 min. The crude mixture was diluted with MilliQ water and filtered with a Millipore membrane . Washings of the solid material were performed with water until the neutrality of the filtrate was achieved, then it was washed once with methanol, and once with diethyl ether. The following day, the powder was dried in an oven (80 °C).First, 25 mg of super-pure SWCNTs  was transferred into 50 mL 8M HNOThe tripeptide L-Leu-D-Phe-D-Phe was synthesized using a chlorotrityl chloride resin and Fmoc-protection strategy, isolated by high-performance liquid chromatography (HPLC) in reverse phase, and identified by nuclear magnetic resonance (NMR) and by electrospray ionization mass spectrometry (ESI-MS) coupled to LC, according to previously published procedures .−1 until the temperature reached 800 °C.TGA was performed using a TGA5500 instrument  with air or nitrogen gas, as indicated further below in N,N-dimethylformamide with <0.1 mg of CNTs. Vis-NIR spectroscopy was performed on a Cary 5000 UV-Vis-NIR instrument, using a rate of scanning of 2400 nm min−1 and a resolution of 4 nm.CNT dispersions were prepared in 1-centimeter quartz cuvettes, using 5 mL −1.Microscopy-grade glass slides were used as the substrate for samples, which were deposited on top of the glass; they were then dried during the night. Raman analysis was performed on ≥5 areas per sample using an inVia 50 instrument from Renishaw  and a 785-nm laser (0.25 mW) scanning every cmhttps://imagej.net).TEM analyses were performed using a JEM 2100  at a voltage of 100 kV. Samples were freshly prepared, then they were carefully placed onto TEM grids (carbon-lacey) that had been treated with UV-Ozone Procleaner Plus for 360 s and dried under vacuum. Negative staining was used for contrast (potassium phosphotungstate at 2% with pH 7.2). Micrograph analysis was carried out with ImageJ2 software from FIJI .FT-IR spectroscopy was performed in attenuated total reflectance (ATR) mode, using a crystal of germanium and applying a resolution of 4 cmFirst, 2.0 mg of ox-CNTs were poured into a solution (1.0 mL) with 5.0 mg of the tripeptide in sodium phosphate buffer  and the dispersion was placed in a sonicator for 15 min. Then, another 1.0 mL of slightly acidic sodium-phosphate buffer  was added to attain a final pH of 7.4, which initiated peptide self-organization into a hydrogel. Samples with 0.1 mg/mL CNTs were prepared accordingly, with a 10-fold reduction in the final amount of CNTs present in the hydrogels.Oscillatory rheology was performed using a Malvern Kinexus Ultra Plus instrument  and a substrate consisting of a plate (20 mm) made of stainless steel and featuring parallel geometry. Other parameters included a gap of 1 mm and an ambient temperature (25 °C) that was maintained with a Peltier system . Hydrogel samples were freshly prepared on the equipment. Kinetics were monitored at 1 Hz and 1 Pa for 60 min, followed by a sweep of frequency from 10 to 0.1 Hz at a stress of 1 Pa. Finally, stress sweeps were recorded from 1 Pa until 100–200 Pa at 1 Hz.SWCNTs and DWCNTs were first oxidized with nitric acid to favor their dispersibility in aqueous buffers compatible with tripeptide self-assembly. Thermogravimetric analysis in air a of the 11OUT). The signals that were present between 1000 and 1370 nm were mainly associated with the corresponding transition of the inner walls of semiconducting DWCNTs (E11IN), as well as to the analogous transition of SWCNTs (E11SW), and to the DWCNT outer walls’ second optical transition (E22OUT). The latter two signals have been reported to be weaker than the first one, and this observation can also be ascribed to lower amounts of SWCNTs. After oxidation, the E11OUT peak was significantly reduced in intensity, while the rest of the spectrum did not change significantly. Considering that the loss of optical response can be ascribed to the grafting of chemical functionalities, we inferred that the oxidation occurred only on DWCNTs’ outer walls while maintaining the integrity of the interior walls. The modest reduction in magnitude noted for the remaining signals was rationalized as being due to the minor contributions of E11SW and E22OUT, which we envisaged would be impacted by oxidation . Th. Th61]. xidation . In agrew/w relative to the L-Leu-D-Phe-D-Phe, the ox-SWCNTs’ tendency to aggregate was remarkably higher than that of ox-DWCNTs. We attributed the difference to the extent of grafting of the polar functional groups on their surface since the length and diameter between the tubes were comparable.The tripeptide L-Leu-D-Phe-D-Phe was produced using a chlorotrityl chloride resin and Fmoc-protection strategy, purified by HPLC in reverse phase, and characterized spectroscopically, as previously described . In the The viscoelastic character of the materials was investigated by rheometry, using oscillation analyses . Time swWithin an hour, the hydrogels with ox-SWCNTs a reachedTEM analyses were performed to shed further light on the composite hydrogels . All samThe design of composite hydrogels based on the supramolecular organization of short peptides and CNMs is not trivial. The effects of changing properties from one CNM to the other are still being elucidated. Specifically, an analogous elongated morphology of CNTs and L-Leu-D-Phe-D-Phe assemblies is useful to improve the viscoelastic properties of these hydrogels. CNT oxidation is key to mitigating their strong tendency toward aggregation. However, striking the right balance is challenging for SWCNTs, since chemical oxidation comes at the expense of their structural integrity, leading to significant sample loss. Conversely, DWCNTs are an attractive alternative type of nanomaterial that allows to introduce polar groups on their outer walls, while preserving the inner walls . Further